The gene regulatory network that controls segmentation in insects has adapted to different developmental situations. For instance, the Bicoid morphogen plays a pivotal role in patterning the anterior of the long germ Drosophila embryo. However, no bicoid homologue has been found outside Diptera, even in other long germ insects. Here, we propose to continue our work on the long germ embryo of Nasonia (Nv), the wasp that is the focus of much of our studies. We have shown that four maternal functions are required to pattern the Nasonia embryo: hunchback (hb), orthodenticle (otd1), giant and caudal are the key maternal components of an ancestral patterning system whose function has been taken over by bicoid in Drosophila. We will ask how these genes act together to pattern the axis and achieve segmentation of the Nasonia embryo. Aim 1. We will continue our study of gap genes in Nasonia. We will test their cross-regulatory interactions and analyze their phenotypes using parental RNAi that works very well in Nasonia. We will pursue our analysis of pair-rule genes, in particular even-skipped (eve) and ftz whose expression patterns are dramatically different from those of flies, in particular in the posterior regions. We will test how they are controlled by upstream factors and will investigate the phenotype of pRNAi embryos. Aim 2 We will study the molecular regulation of pair-rule genes, focusing on eve whose control is understood in exquisite detail in Drosophila. We will identify the individual regulatory modules that control anterior pair-rule stripes of Nv eve, or posterior segmental expression. This will highlight the two modes of segmentation that appear to co-exist in insects: one for the anterior (and the only one in Drosophila), and one for the posterior segments that is reminiscent of the mode of segmentation of Tribolium. We will analyze the molecular mechanisms of Nv eve stripe 2 expression using genetics, bioinformatics and transgenics. Site-directed mutagenesis of the eve regulatory region will allow us to assess the roles of Otd1, Hb, gap gene as well as pair-rule gene products and how they differ from their roles in Drosophila. Aim 3 Our preliminary data indicate that localization of mRNA is a critical component of Nasonia's ability to pattern its embryo in the absence of bicoid. Nv otd1 mRNA is localized at both poles while giant is present only at the anterior and caudal form a mRNA gradient. We will evaluate the ability of the 3'UTR of these genes to direct mRNA localization in Drosophila and in Nasonia and assess whether the signals work across species. We will manipulate the cytoskeleton to define the requirements for mRNA localization machinery. The function of genes involved in this process in Drosophila will be tested by RNAi in Nasonia, and their importance for localizing each mRNA determined. PUBLIC HEALTH RELEVANCE: The Drosophila embryo is the best known complex biological system. However, it is a unique example that does not represent the diversity of life. By comparing Drosophila development with Nasonia, we will be able to distinguish general mechanisms that have broad implications for the development of animals from more specific processes.